EP1512753B1 - Reagens zur Detektion des thermostable direct hemolysin-related Hemolysin Gens von vibrio parahaemolyticus - Google Patents

Reagens zur Detektion des thermostable direct hemolysin-related Hemolysin Gens von vibrio parahaemolyticus Download PDF

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EP1512753B1
EP1512753B1 EP04020602A EP04020602A EP1512753B1 EP 1512753 B1 EP1512753 B1 EP 1512753B1 EP 04020602 A EP04020602 A EP 04020602A EP 04020602 A EP04020602 A EP 04020602A EP 1512753 B1 EP1512753 B1 EP 1512753B1
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rna
sequence
primer
dna
oligonucleotide
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EP1512753A1 (de
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Noriyoshi Masuda
Ryuichi Horie
Kiyoshi Yasukawa
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Tosoh Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/173—Nucleic acid detection characterized by the use of physical, structural and functional properties staining/intercalating agent, e.g. ethidium bromide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a detection reagent for detecting Vibrio parahaemolyticus in clinical examinations, public health examinations, food evaluations and food poisoning examinations.
  • Vibrio parahaemolyticus is commonly known as a causative organism of infectious food poisoning. In contrast to 95% or more of Vibrio parahaemolyticus isolated from gastroenteritis patients being Kanagawa phenomenon-positive bacteria demonstrating hemolytic activity in Wagatsuma medium, 99% of the bacteria isolated from fish and water are Kanagawa phenomenon-negative. Thus, there is considered to be a close relationship between pathogenic Vibrio parahaemolyticus and the Kanagawa phenomenon.
  • TDH thermostable direct hemolysin
  • TRH TDH-related hemolysin
  • Vibrio parahaemolyticus comprising evaluation for Kanagawa phenomenon following enrichment culturing or isolation culturing are known, methods which detect a specific sequence present in the Vibrio parahaemolyticus gene or an RNA derived from said gene following the amplification of such a sequence are preferable in terms of sensitivity, speed and ease of procedure.
  • a method that amplifies a target nucleic acid at a constant temperature is particularly preferable in terms of automation of a testing system.
  • an RNA amplification process in which a double-strand RNA-DNA is formed by producing a cDNA with an RNA-dependent DNA polymerase using a specific sequence of an RNA derived from said TRH1 or TRH2 gene as a template, as well as a first primer having a sequence complementary to said specific sequence, and a second primer having a sequence homologous to said specific sequence, wherein the first primer or second primer has a sequence in which a promoter sequence of an RNA polymerase is added to the 5' end of one of the primers, degrading the RNA of the double-strand RNA-DNA by ribonuclease H, thereby producing a single-strand DNA, and producing a double-strand DNA having the promoter sequence capable of transcribing the RNA composed of the RNA sequence or the sequence complementary to the RNA sequence with a DNA-dependent DNA polymerase using said single-strand DNA as a template, wherein said double-strand DNA produces an RNA transcription product in the presence of the
  • the aforementioned method has the following problems. First, it has low sensitivity. According to Japanese Unexamined Patent Publication No. 2001-340087, data is only indicated for the detection of TRH1 RNA for which the initial RNA amount is at least 10 3 copies, and it is unclear as to whether TRH1 RNA can be detected if the initial RNA amount is less than 10 3 copies (e.g., 10 2 copies). According to Japanese Unexamined Patent Publication No. 2001-340088, data is only indicated for the detection or TRH2 RNA for which the initial RNA amount is at least 10 3 copies, and it is unclear as to whether TRH2 RNA can be detected if the initial RNA amount is less than 10 3 copies (e.g., 10 2 copies).
  • the second problem is that any reagent capable of detecting both TRH1 and TRH2, which is required for practical use, was not reported.
  • the reagent capable of detecting TRH1 RNA at an initial RNA amount of at least 10 3 copies does not detect TRH2.
  • the reagent capable of detecting TRH2 RNA at an initial RNA amount of at least 10 3 copies does not detect TRH1.
  • the object of the present invention is to provide a detection reagent for TRH RNA that has superior sensitivity and speed, and detects both TRH1 and TRH2 of Vibrio parahaemolyticus.
  • thermostable direct hemolysin-related hemolysin (TRH) RNA of Vibrio parahaemolyticus having a superior sensitivity and a higher speed
  • TRH thermostable direct hemolysin-related hemolysin
  • the present invention relates to a detection reagent for use in detecting the TRH gene of Vibrio parahaemolyticus present in a sample that is used in a detection method using an RNA amplification process comprising the steps of:
  • the highly stringent condition refers to hybridization conditions and, for example, those indicated in the following examples consisting of carrying out a hybridization at a temperature of 44°C in the presence of 60 mM Tris, 17 mM magnesium chloride, 100 to 130 mM potassium chloride and 1 mM DTT.
  • the aforementioned first primer may be an oligonucleotide consisting of the sequence listed as SEQ. ID No. 2
  • the aforementioned second primer may be an oligonucleotide consisting of the sequence listed as SEQ. ID No. 1.
  • an oligonucleotide having a sequence complementary to the aforementioned first primer with its sequence from the 5' end to the 3' end being reversed should be used as the first primer and an oligonucleotide having a sequence complementary to the aforementioned second primer with its sequence from the 5' end to the 3' end being reversed should be used as the second primer.
  • the aforementioned RNA amplification process is carried out in the presence of a cleaving oligonucleotide that cleaves the aforementioned target RNA at the 5' end of the aforementioned specific sequence and has a sequence complementary to the region adjacent to and overlapping with the 5' end of said specific sequence.
  • Said oligonucleotide is preferably an oligonucleotide consisting of the sequence listed as SEQ. ID No. 4, 5 or 6.
  • the aforementioned RNA amplification process is carried out in the presence of an oligonucleotide labeled with an intercalator fluorescent pigment, and the detection of the thermostable direct hemolysin-related hemolysin of Vibrio parahaemolyticus is carried out by measuring the fluorescent intensity of the reaction solution.
  • the sequence of said oligonucleotide is complementary to at least a portion of the sequence of the mRNA transcription product, and in the situation where complementary binding of said oligonucleotide to said RNA transcription product occurs, the fluorescent properties of the reaction solution change in comparison with the situation where no complex is formed.
  • the aforementioned oligonucleotide consists of at least 10 contiguous bases in any of the sequences listed as SEQ. ID No. 3.
  • the entire length of the base sequences listed in each of the sequence listings can be used for the first and second primers, respectively, as about 10 bases are sufficient for specific binding to a specific nucleic acid sequence or the like, a combination of at least 10 contiguous bases in each sequence may also be used.
  • the amplification process of the present invention includes the NASBA method, 3SR method or, for example, the RNA detection method (TRC method) described in Japanese Unexamined Patent Publication No. 2000-014400, which amplifies TRH1 and TRH2 RNA by concerted action of reverse transcriptase and RNA polymerase (by reacting them under a condition where the reverse transcriptase and RNA polymerase act in concert).
  • TRC method RNA detection method described in Japanese Unexamined Patent Publication No. 2000-014400
  • temperature it is preferably 35 to 50°C.
  • a preferable method for cleaving the target RNA in this manner preferably consists of cleaving the target RNA with ribonuclease H, or the like, by adding an oligonucleotide having a sequence complementary to the region adjacent to and overlapping with the 5' end of the specific sequence (cleaving oligonucleotide).
  • Said oligonucleotide is preferably an oligonucleotide consisting of a sequence listed as SEQ. ID No. 4, 5 or 6.
  • the 3' end hydroxyl group is preferably chemically modified, for example, aminated, in order to suppress an elongation reaction from the 3' end.
  • the aforementioned nucleic acid amplification is preferably carried out in the presence of an oligonucleotide labeled with an intercalator fluorescent pigment followed by measurement of the change in the fluorescent properties of the reaction solution.
  • the intercalator fluorescent pigment is bound to the phosphorous atom in the oligonucleotide by means of a linker
  • the intercalator portion that forms a double strand with the target nucleic acid intercalates to the double strand portion resulting in a change in fluorescent properties, thereby resulting in the characteristic of not requiring separation and analysis (Ishiguro, T. et al. (1996) Nucleic Acid Res. 24 (24) 4992-4997).
  • the sequence bound by the said oligonucleotide may be any sequence specific for TRH RNA and, although there are no particular limitations thereon, a sequence consisting of at least 10 contiguous bases in the sequence listed as SEQ. ID No. 3 or its complementary sequence is preferable.
  • the hydroxyl group at the 3' end of said oligonucleotide is preferably chemically modified (such as by addition of glycolic acid) to suppress the elongation reaction which may occur by using this oligonucleotide as a primer.
  • TRH1 RNA and TRH2 RNA of Vibrio parahaemolyticus can be amplified and detected in a single tube, at a constant temperature and in a single step both rapidly and with high sensitivity, thereby facilitating application to automation.
  • TRH RNA of Vibrio parahaemolyticus was compared for combinations (a) through (j) shown in Table 1 and Fig. 1.
  • Table 1 shows the combinations of the first primer, the second primer and the cleaving oligonucleotide used in the experimental system along with the lengths of the specific bands amplified using those combinations.
  • the locations and amplified regions of the oligonucleotides in TRH RNA of Vibrio parahaemolyticus are shown for each of the oligonucleotide combinations in Fig. 1.
  • the hydroxyl group on the 3' end of the base sequence of the cleaving oligonucleotide was aminated.
  • the region from the 1st "A” to the 22nd “A” from the 5' end of the base sequence of the second primer is a T7 promoter region, and the subsequent region of from the 23rd “G” to the 28th "A” is an enhancer sequence.
  • Table 2 shows the results of measuring TRH1 and TRH2 RNA at 10 3 copies/test using the combinations of oligonucleotides shown in Table 1. All of the combinations of oligonucleotides shown in Table 1 detected TRH1 and TRH2 RNA within 15 minutes.
  • the detection method of the invention of the present application is useful for detecting TRH1 and TRH2 RNA of Vibrio parahaemolyticus both simultaneously and with high sensitivity.
  • the oligonucleotide of the invention of the present application is not limited to that of the base sequences listed in the sequence listings (having 22 to 30 bases), but rather can be a nucleotide comprised of at least 10 contiguous bases in those sequences. This is because it is clear that a base sequence of about 10 bases is sufficient for ensuring specificity to a target nucleic acid of a primer or probe under comparative low-temperature (preferably 44°C) conditions.

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Claims (9)

  1. Detektionsreagens zur Verwendung beim Detektieren der in einer Probe vorliegenden, beiden thermostabilen direkt Hämolysin-bezogenen Hämolysin (TRH1 und TRH2)-Gene von Vibrio parahaemolyticus, das in einem Detektionsverfahren verwendet wird, welches einen RNA-Amplifikationsprozess verwendet, der folgende Schritte umfasst:
    Produzieren einer cDNA mit einer RNA-abhängigen DNA-Polymerase unter Verwendung einer spezifischen Sequenz einer RNA, die aus den TRH1- und TRH2-Genen abgeleitet wurde, als Matrize, sowie eines ersten Primers, der eine zu der spezifischen Sequenz komplementäre Sequenz aufweist, und eines zweiten Primers, der eine zu der spezifischen Sequenz homologe Sequenz aufweist, wodurch eine doppelsträngige RNA-DNA gebildet wird, wobei entweder der erste Primer oder der zweite Primer eine Sequenz aufweist, in der eine Promotorsequenz einer RNA-Polymerase an ihrem 5'-Ende angelagert wurde;
    Degradieren des RNA-Abschnitts der doppelsträngigen RNA-DNA durch Ribonuklease H, wodurch eine einzelsträngige DNA produziert wird; und
    Produzieren einer doppelsträngigen DNA, welche die Promotorsequenz aufweist, die in der Lage ist, die sich aus der spezifischen Sequenz der RNA oder der zu der spezifischen Sequenz der RNA komplementären Sequenz zusammensetzende RNA mit einer DNA-abhängigen DNA-Polymerase unter Verwendung der einzelsträngigen DNA als Matrize zu transkribieren; wobei
    die doppelsträngige DNA ein RNA-Transkriptionsprodukt in Anwesenheit der RNA-Polymerase produziert, und das RNA-Transkriptionsprodukt als Matrize für die nachfolgende cDNA-Synthese mit der RNA-abhängigen DNA-Polymerase dient;
    wobei das Reagens umfasst:
    als den ersten Primer ein Oligonukleotid, das aus mindestens 10 zusammenhängenden Basen der als SEQ. ID Nr. 2 aufgelisteten Sequenz besteht und in der Lage ist, sich genau an die spezifische Sequenz zu binden; und
    als den zweiten Primer ein Oligonukleotid, das aus mindestens 10 zusammenhängenden Basen der als SEQ. ID Nr. 1 aufgelisteten Sequenz besteht und in der Lage ist, sich genau an die zu der spezifischen Sequenz komplementäre Sequenz zu binden.
  2. Detektionsreagens nach Anspruch 1, wobei der erste Primer ein aus der als SEQ. ID Nr. 2 aufgelisteten Sequenz bestehendes Oligonukleotid ist.
  3. Detektionsreagens nach Anspruch 1 oder 2, wobei der zweite Primer ein aus der als SEQ. ID Nr. 1 aufgelisteten Sequenz bestehendes Oligonukleotid ist.
  4. Detektionsverfahren zum Detektieren der in einer Probe vorliegenden, beiden thermostabilen direkt Hämolysin-bezogenen Hämolysin (TRH1- und TRH2)-Gene von Vibrio parahaemolyticus, das einen RNA-Amplifikationsprozess verwendet, der folgende Schritte umfasst:
    Produzieren einer cDNA mit einer RNA-abhängigen DNA-Polymerase, unter Verwendung einer spezifischen Sequenz einer RNA, die aus den TRH1- und TRH2-Genen abgeleitet wurde, als Matrize, sowie eines ersten Primers, der eine zu der spezifischen Sequenz komplementäre Sequenz aufweist, und eines zweiten Primers, der eine zu der spezifischen Sequenz homologe Sequenz aufweist, wodurch eine doppelsträngige RNA-DNA gebildet wird, wobei entweder der erste Primer oder der zweite Primer eine Sequenz aufweist, in der eine Promotorsequenz einer RNA-Polymerase an ihrem 5'-Ende angelagert wurde;
    Degradieren des RNA-Abschnitts der doppelsträngigen RNA-DNA durch Ribonuklease H, wodurch eine einzelsträngige DNA produziert wird; und
    Produzieren einer doppelsträngigen DNA, welche die Promotorsequenz aufweist, die in der Lage ist, die sich aus der spezifischen Sequenz der RNA oder der zu der spezifischen Sequenz der RNA komplementären Sequenz zusammensetzende RNA mit einer DNA-abhängigen DNA-Polymerase unter Verwendung der einzelsträngigen DNA als Matrize zu transkribieren; wobei
    die doppelsträngige DNA ein RNA-Transkriptionsprodukt in Anwesenheit der RNA-Polymerase produziert, und das RNA-Transkriptionsprodukt als Matrize für die nachfolgende cDNA-Synthese mit der RNA-abhängigen DNA-Polymerase dient; und
    wobei
    der erste Primer ein aus mindestens 10 zusammenhängenden Basen der als SEQ. ID Nr. 2 aufgelisteten Sequenz bestehendes Oligonukleotid ist und in der Lage ist, sich genau an die spezifische Sequenz zu binden, und
    der zweite Primer, ein aus mindestens 10 zusammenhängenden Basen der als SEQ. ID Nr. 1 aufgelisteten Sequenz bestehendes Oligonukleotid ist und in der Lage ist, sich genau an die zu der spezifischen Sequenz komplementäre Sequenz zu binden.
  5. Detektionsverfahren nach Anspruch 4, wobei der erste Primer ein aus der als SEQ. ID Nr. 2 aufgelisteten Sequenz bestehendes Oligonukleotid ist.
  6. Detektionsverfahren nach Anspruch 4 oder 5, wobei der zweite Primer ein aus der als SEQ. ID Nr. 1 aufgelisteten Sequenz bestehendes Oligonukleotid ist.
  7. Detektionsverfahren nach einem der Ansprüche 4 bis 6, wobei der RNA-Amplifikationsprozess in Anwesenheit eines spaltenden Oligonukleotids durchgeführt wird, das die Ziel-RNA am 5'-Ende der spezifischen Sequenz spaltet und eine Sequenz aufweist, die zu der Region komplementär ist, die an das 5'-Ende der spezifischen Sequenz angrenzt und mit diesem überlappt;
    wobei das Oligonukleotid ein aus der als SEQ. ID Nr. 4, 5 oder 6 aufgelisteten Sequenz bestehendes Oligonukleotid ist.
  8. Detektionsverfahren nach einem der Ansprüche 4 bis 7, wobei der RNA-Amplifikationsprozess in Anwesenheit eines mit einem Interkalator-Fluoreszenzpigment markierten Oligonukleotids durchgeführt wird, und die Detektion des thermostabilen direkt Hämolysin-bezogenen Hämolysin von Vibrio parahaemolyticus durch Messen der Fluoreszenzintensität der Reaktionslösung durchgeführt wird; wobei die Sequenz des Oligonukleotids zu mindestens einem Abschnitt der Sequenz des mRNA-Transkriptionsprodukts komplementär ist, und sich die Fluoreszenzeigenschaften der Reaktionslösung in einer Situation, wo die komplementäre Bindung des Oligonukleotids an das RNA-Transkriptionsprodukt erfolgt, im Vergleich zu der Situation, wo kein Komplex gebildet wird, ändern.
  9. Detektionsverfahren nach Anspruch 8, wobei das mit dem Interkalator-Fluoreszenzpigment markierte Oligonukleotid aus mindestens 10 zusammenhängenden Basen der als SEQ. ID Nr. 3 aufgelisteten Sequenz besteht.
EP04020602A 2003-09-05 2004-08-31 Reagens zur Detektion des thermostable direct hemolysin-related Hemolysin Gens von vibrio parahaemolyticus Expired - Lifetime EP1512753B1 (de)

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JP2003314142 2003-09-05
JP2003314142A JP2005080531A (ja) 2003-09-05 2003-09-05 腸炎ビブリオ菌の耐熱性溶血毒類似溶血毒素遺伝子の検出試薬

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CN102676459B (zh) * 2011-03-09 2014-04-02 上海海洋大学 一种抗副溶血弧菌耐热溶血毒素单克隆抗体及其制备方法
WO2014039037A1 (en) * 2012-09-06 2014-03-13 University Of South Carolina Pcr primers for detection of vibrio parahaemolyticus thermostable direct hemolysin (tdh) and tdh-related hemolysin genes
WO2015190106A1 (ja) * 2014-06-11 2015-12-17 東洋製罐グループホールディングス株式会社 食中毒菌検出用担体、食中毒菌検出用キット、食中毒菌の検出方法、及び食中毒菌用ｐｃｒ反応液
JP2016000015A (ja) * 2014-06-11 2016-01-07 東洋製罐グループホールディングス株式会社 食中毒菌検出用担体、及び食中毒菌検出用キット
JP2016000016A (ja) * 2014-06-11 2016-01-07 東洋製罐グループホールディングス株式会社 食中毒菌の検出方法、及び食中毒菌用ｐｃｒ反応液
CN111411161B (zh) * 2020-04-02 2021-02-23 深圳市疾病预防控制中心 检测副溶血弧菌k抗原基因分型的引物组和试剂盒
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KR20050027011A (ko) 2005-03-17
US20050112628A1 (en) 2005-05-26
CN1637152B (zh) 2010-09-01
CN1637152A (zh) 2005-07-13
EP1512753A1 (de) 2005-03-09
JP2005080531A (ja) 2005-03-31
DE602004005936T2 (de) 2007-09-06
US7495094B2 (en) 2009-02-24

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